Erratum in

J Biol Chem. 2015 Jan 23;290(4):2301.

Abstract

The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK.

NPC is essential for growth upon osmostress. Mutations on specific NPC components render cells osmosensitive. Wild type (wt) and the indicated mutant strains were grown to mid-log exponential phase and spotted, making serial dilutions in YPD plates without or with 1.2 m NaCl or 2 m sorbitol. Growth at 30 °C was scored after 3 days.

NPC is essential for gene expression upon osmostress. WT and the indicated viable NPC mutant strains were grown to mid-log phase in rich medium and subjected to 0.4 m NaCl or 1.2 m NaCl osmostress for the indicated lengths of time. Total RNA was extracted and blotted against STL1 and CTT1 and RDN18 as the loading control.

NPC is essential for gene expression upon osmostress. WT and the indicated strains were grown to mid-log phase in rich medium and then subjected to osmostress (1 m NaCl) for the indicated length of time. Total RNA was extracted and assayed by Northern blot for transcript levels of STL1, CTT1, and GRE2 and RDN18 as the loading control.

Localization of Hog1 or transcription factors Hot1, Msn2, and Sko1 is not altered in NPC mutant strains.A, Hog1 localization was not affected in NPC mutant strains. WT and the indicated NPC mutant strains expressing Hog1-GFP were grown in SD medium to mid-log phase and subjected to osmostress (0.4 m NaCl, 5 min). DAPI staining to reveal nuclei and epifluorescence pictures were taken without (basal conditions) and with stress. The percentage of cells with Hog1 nuclear accumulation upon stress is shown in the lower panel; data are the mean and S.D. of three independent experiments. B, Hot1, Msn2, and Sko1 localization is not affected in NPC mutant strains. WT and the indicated NPC mutant strains expressing Hot1-GFP, Mns2-GFP, and Sko1-GFP were grown in SD medium to mid-log phase and subjected to osmostress (0.4 m NaCl, 5 min). Nomarsky and epifluorescence pictures were taken without (−) and with stress (+). DAPI staining to reveal nuclei in the wild type strain upon stress is shown in the lower panel.

Hog1 co-precipitates with the NPC and phosphorylates some of its components.A, in vivo binding of Hog1 and the NPC is shown. TAP-tagged Nup188, Nup84, Nup82, Nup60, or Nup2 strains expressing GST or GST-Hog1 were grown to mid-log phase and subjected to brief osmotic shock (0.4 m NaCl, 10 min). GST proteins were pulled down by glutathione-Sepharose 4B, and the presence of TAP-tagged proteins was probed by immunoblotting using anti-TAP (PAP; Sigma) (upper panel). Total extract represents <20% of total input protein (middle panel). Amounts of precipitated (prec.) GST proteins were detected using anti-GST (lower panel). B, GST-Hog1 and Nup2-HA or Nup60-HA reciprocal immunoprecipitations are shown. Nup2-HA and Nup60-HA co-immunoprecipitates with GST-Hog1 in a GST pulldown assay (left panels). Cse2-HA was added as negative control. Correspondingly, Hog1 co-immunoprecipitates with Nup2-HA or Nup60-HA in a HA pulldown assay (right panels). C, in vitro phosphorylation of Nup2, Nup60, and Nup1 by Hog1 is shown. Recombinant GST-fused proteins from E. coli were phosphorylated in vitro by Hog1. A non-relevant protein (GST-tagged Rpn2) was added as a control to exclude possible nonspecific phosphorylation of Nups by Hog1. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography (upper panel). Total protein levels were detected by staining with Coomassie Brilliant Blue (lower panel).

nup2 and nup60 mutants display defects in STL1 mRNA export upon osmostress.A, the localization of STL1 RNA upon stress (0.4 m NaCl; upper panels) and in basal conditions (lower panel) was assessed by in situ hybridization using Cy3-labbeled probes against STL1. DNA was stained with DAPI. A merged zoom image is shown in the right panels. B, the percentage of cells with impaired export of STL1 mRNA in response to osmostress is shown. A temperature-sensitive allele of MEX67 (mex67-5) was used as a positive control of impaired total mRNA export. Data are the mean and S.D. of three independent experiments.

Nuclear basket proteins bind to stress-dependent genes through Hog1 upon osmostress. WT and hog1 strains expressing HA-tagged Nup60, Nup1, and Nup2 were grown to mid-log phase and subjected (open bars) to osmostress (0.4 m NaCl, 10 min) or not (filled bars). Proteins were immunoprecipitated using anti-HA monoclonal antibody, and binding to the promoter (left panels) and ORF (right panels) regions of STL1 and CTT1 loci was analyzed. The real-time PCR results are shown as -fold induction of treated relative to non-treated (time 0) samples normalized to a telomere internal control. Data are the mean and S.D. of three independent experiments. The statistical significance of the difference was assessed by a paired Student's t test of acceptance of equality at (p value < 0.05) comparing the Nup binding of a wild type versus hog1 upon stress.

NPC localization is not affected upon osmostress. Yeast strains expressing Nup60-GFP, Nup1-GFP, or Nup2-GFP were grown in SD medium to mid-log phase and subjected to osmostress (0.4 m NaCl or 1.2 m NaCl, 5 min). Epifluorescence pictures were taken without (basal conditions) and with stress.

A, Nup1 and Nup2 are phosphorylated in vitro by Hog1. Recombinant GST-fused wild type and mutant proteins were purified from E. coli, and in vitro kinase assays were performed as in B. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography (upper panel). GST-tagged Nup proteins were detected by staining with Coomassie Brilliant Blue (lower panel). B, shown is -fold change in phosphorylation upon 0.4 m NaCl treatment (5 min) at the indicated residues in Nup2 in wild type (blue) and hog1 mutant (red) strains. Ratios have been calculated from absolute phosphorylation intensities of unstressed and osmostressed wild type and hog1 mutant samples. The table shows the Nup2 residues that have been found to be phosphorylated in this study (first column), the position of the phosphorylated residue (red) in the MS-detected phosphopeptide (second column), and ratios of wild type stressed/wild type unstressed and hog1 mutant stressed/hog1 mutant unstressed (third and fourth column, respectively).